Functional polymorphism in the key members of the Toll signalling pathway in the population of the Czech Red Pied (Czech Simmental) cattle was screened and interpreted. Focus was on the transcription factor NFkappaB as the main pleiotropic factor for phenotypic traits and on MyD88 as an interactor insufficiently studied until now. Hybrid resequencing with Illumina X-Ten WGS and PacBio amplicon sequencing yielded 22 and 13 SNPs in NFkappaB1 and NFkappaB2 genes, respectively. Over 30 SNPs were found in the MYD88 gene.